Walnut protein peptide with ADH and ALDH activation activity and screening method thereof

Abstract

The invention discloses a walnut protein peptide with ADH and ALDH activation activity and a screening method thereof, and belongs to the technical field of food-borne bioactive peptide development. The method for rapidly screening ADH and ALDH activating peptides is constructed by creatively combining polypeptiomics, various screening conditions and molecular docking, and the defect that time and labor are consumed due to step-by-step separation and purification is overcome. According to the invention, the high-activity ADH and ALDH activated polypeptide-PGHFEAF is identified from walnut protein for the first time, the polypeptide can resist gastrointestinal tract digestion, the butt joint binding energy of the polypeptide with ADH and ALDH is-8.2 kcal / mol and-10.5 kcal / mol respectively, the ADH activated EC50 value is 0.49 + / -0.09 mM, and the ALDH activated EC50 value is 1.85 + / -0.28 mM.

Description

Technical Field

[0001] The invention relates to a walnut protein peptide with ADH and ALDH activation activity and a screening method thereof, belonging to the technical field of development of food-derived bioactive peptides. Background Art

[0002] Drinking has become a common phenomenon worldwide, but excessive drinking seriously affects human health. The World Health Organization's global status report on alcohol and health pointed out that about 3 million people die from drinking every year, accounting for 5.3% of all deaths, and more than 60 diseases are related to long-term excessive drinking. Alcohol causes varying degrees of harm to various organs of the human body, among which the brain and liver are the most harmful, and the most representative ones are fatty liver, hepatitis and cirrhosis. In many countries, there is a linear correlation between alcohol intake and mortality from liver disease. So far, people have not been able to obtain satisfactory therapeutic drugs. Although some drugs are effective in promoting alcohol metabolism and improving liver conditions, they may also cause varying degrees of side effects and physiological toxicity. Therefore, finding effective and safe functional substances for sobering up and treating alcoholic liver damage has important research significance and broad development prospects.

[0003] Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are the main enzymes that catalyze the oxidation metabolism of alcohol in the liver. 80-90% of alcohol is converted into acetaldehyde under the action of ADH, and acetaldehyde is then catalyzed and oxidized by ALDH to produce non-toxic acetic acid. Acetic acid enters the blood circulation and participates in the tricarboxylic acid cycle in organs such as the kidneys, and is finally metabolized to produce CO 2 and H 2 O. ADH, as the first limiting factor of alcohol metabolism in the body, plays an important role in limiting the metabolic rate of alcohol; and ALDH, as the main substance that catalyzes the metabolism of the toxic substance acetaldehyde, can slow down the damage of acetaldehyde to the human body. When the alcohol intake is low, ADH is the main metabolic pathway of alcohol. When drinking excessively, it will inhibit the oxidation pathway in which ADH participates, and highly induce the expression and secretion of CYP2E1. The CYP2E1 pathway produces a large number of reactive oxygen free radicals (ROS), which is one of the main causes of liver damage. Therefore, by activating the activity of ADH, inhibiting the expression of the CYP2E1 pathway, reducing the generation of reactive oxygen free radicals, increasing the activity of ALDH, accelerating the conversion of acetaldehyde into non-toxic acetic acid, and reducing the accumulation of acetaldehyde in the liver, alcoholic liver damage can be improved to a certain extent.

[0004] In recent years, the activity of bioactive peptides in the field of sobering up and protecting the liver has gradually become a research hotspot. However, there is a relative lack of identification of peptide sequences with sobering up and protecting the liver and related research, including the elucidation of the molecular mechanism of peptide activation of ADH and ALDH. Therefore, it is urgent to develop a fast, convenient and efficient screening method for sobering up and protecting the liver. Walnut protein is a protein resource with rich nutrition, balanced amino acid composition and high bioavailability. So far, no reports on ADH and ALDH activating peptides from walnut protein have been found. Summary of the invention

[0005] The purpose of the present invention is to provide a peptide segment with the ability to activate ADH and ALDH, which can be used for the application and development of sobering up and liver protecting products.

[0006] The present invention provides a method for screening ADH and ALDH activating peptides from walnut protein, which mainly comprises the following steps:

[0007] (1) Extracting walnut protein from walnut kernels using alkali dissolution and acid precipitation

[0008] Walnut protein powder and water were mixed at a ratio of 1:20 (w:v), the pH was adjusted to 10.00-11.0, and the mixture was stirred continuously for 3-4 hours at room temperature and then centrifuged (7000r / min, 15-20min), the supernatant was collected and stored, the pH of the supernatant was adjusted to 4.5 and then centrifuged again (7000r / min, 15-20min), the precipitate was redissolved, and the pH was adjusted to 7.0. After dialysis, the mixture was freeze-dried to obtain walnut protein powder.

[0009] (2) Producing walnut peptides by hydrolyzing walnut protein with protease

[0010] The walnut protein powder obtained in step (1) is mixed with water in a certain proportion, and then a protease is added to hydrolyze the walnut protein. After the hydrolysis is completed, the walnut protein hydrolyzate is inactivated by boiling water, cooled to room temperature, the pH is adjusted to 7.0, centrifuged, and the supernatant is freeze-dried to obtain a walnut protein hydrolyzate powder, which is stored at -20°C for later use. The protease is: alkaline protease, neutral protease, composite protease and flavor protease.

[0011] (3) In vitro simulation of gastrointestinal digestion

[0012] The walnut peptide obtained in step (2) was mixed with pepsin hydrochloric acid solution (2000U / mL, pH 3.0) in a certain proportion, and the mixture was cultured at a constant temperature (37°C, 150rpm, 2h) to simulate gastric digestion (SGD). After the pH of the digestion system was adjusted to 7.0, the enzyme was rapidly inactivated and cooled, which was recorded as 2h. 100U / mL trypsin was added to the remaining sample to simulate the intestinal digestion (SID) stage. Samples were taken at different time points, the enzyme was inactivated, and the mixture was rapidly cooled.

[0013] (4) Identification and screening

[0014] The peptide sequences of the walnut protein hydrolysate obtained in step (2) are identified by peptideomics, and potential new ADH and ALDH activating peptides are screened out from the walnut peptides using a variety of screening conditions.

[0015] (5) Synthetic Verification

[0016] Molecular docking was used to explore its intrinsic mechanism of activating ADH and ALDH, and the target sequence was synthesized in vitro and its ADH and ALDH activation rates were detected.

[0017] In one embodiment, in step (1), the walnut protein powder is a dry protein powder after fat is removed.

[0018] In one embodiment, in step (2), when the protease is an alkaline protease, the hydrolysis conditions are: temperature of 55°C, pH 8.5, and hydrolysis time of 4 hours; when the protease is a neutral protease, the hydrolysis conditions are: temperature of 45°C, pH 7.0, and hydrolysis time of 4 hours; when the protease is a composite protease, the hydrolysis conditions are: temperature of 55°C, pH 8.0, and hydrolysis time of 3 hours; when the protease is a flavor protease, the hydrolysis conditions are: temperature of 50°C, pH 7.0, and hydrolysis time of 2.5 hours.

[0019] In one embodiment, in step (1), the following implementation conditions are adopted: after walnut protein powder and water are mixed in a ratio of 1:20 (w / v), the pH is adjusted to 10.00-11.0, and centrifuged (7000r / min, 15min) after continuous stirring for 3h at room temperature, the supernatant is collected and stored, and the supernatant pH is adjusted to 4.5 and then centrifuged again (7000r / min, 15min), the precipitate is redissolved, and the pH is adjusted to 7.0. Walnut protein powder is obtained by freeze drying after dialysis. During the hydrolysis process, 1mol / L NaOH or HCl is used to maintain the pH value of the solution constant.

[0020] In one embodiment, in step (2), the following implementation conditions are adopted: walnut protein powder is mixed with water in a ratio of 1:25 (w:v) to prepare a protein solution with a substrate concentration of 4%. After mixing, the pH and temperature are adjusted to the optimal pH and temperature conditions of the enzyme used, and protease is added according to the addition amount of 10000U / g protein of the enzyme substrate ratio for hydrolysis, and stirred for extraction at room temperature for 2-4h. After hydrolysis to a constant pH, the enzyme is inactivated in a boiling water bath for 10min, and after cooling to room temperature, the pH is adjusted to 7.0, and centrifuged at 8000r / min for 20min. The supernatant is collected, dialyzed, and freeze-dried to obtain walnut protein hydrolyzed powder under different protease hydrolysis conditions.

[0021] In one embodiment, in step (3), the following implementation conditions are adopted: weigh 1g of walnut peptide protein hydrolysate in 10mL of 2000U / mL pepsin hydrochloric acid solution (pH3.0), and culture at a constant temperature (37°C, 150rpm, 2h) to simulate gastric digestion (SGD). Then enter the simulated intestinal digestion (SID) stage, adjust the pH of the digestion system to 7.0 and adjust the volume to 20mL, take a sample of 10mL, and then quickly inactivate the enzyme and cool it, which is recorded as 2h. Add 100U / mL trypsin to the remaining sample, take samples at different time points, inactivate the enzyme and cool it quickly. The freeze-dried sample was tested for ADH and ALDH activation activity.

[0022] In one embodiment, step (4) may specifically be performed as follows:

[0023] ① Preliminary screening conditions: The amino acid sequences obtained by sequencing were used for potential biological activity prediction, protein source prediction, toxicity and sensitization prediction by Peptide Ranker software, protein database, Toxinpred software and AllerTOP software. From the sequenced peptides, peptides that are of protein origin, not modified by functional groups, with a peak area of ​​>1.00×106, peptide sequences containing 3-12 amino acids, and activity scores ≥0.7 were selected.

[0024] ② Rapid screening conditions: select peptide sequences with amphiphilicity, steric hindrance ≤0.65, hydrophobic amino acids ≥50% and characteristic amino acids ≥50%. Peptides with such characteristics have potential ADH activation activity.

[0025] In one embodiment, step (5) may specifically adopt the following steps:

[0026] ① Obtain small molecule ligands: Use Chem Draw (version ChemBio Draw 22.0.0) and Chem3D (version ChemBio Draw 22.0.0) software to construct the 2D and 3D structures of the peptide sequences to be docked.

[0027] ② Preparation of PDB files for ADH and ALDH: The protein crystal structures of ADH and ALDH (5ENV and 1O04, respectively) were retrieved from the protein database, and redundant ligands and water molecules were removed for subsequent docking with peptide segments.

[0028] ③Molecular docking: The format of ligand and receptor was converted to PDBQT in AutoDock Tools 1.5.7, and the molecular docking simulation experiment was performed using AutoDockVina software. The result was output as the predicted binding energy. The lower the binding energy, the stronger the binding between the ligand and the protein pocket. The ADH and ALDH activation peptides with published sequences in the prior art were excluded, and the remaining peptides were identified as potential ADH and ALDH activation peptides, which were synthesized in vitro by solid phase synthesis and their activation activity was verified.

[0029] ④ The screened peptides are synthesized by solid phase synthesis in the peptide chemical synthesis method, and the ADH and ALDH activation activities are determined by substrate chemical method to verify the screening results.

[0030] The present invention provides a polypeptide PGHFEAF, the amino acid sequence of the polypeptide is

[0031] Pro-Gly-His-Phe-Glu-Ala-Phe.

[0032] The present invention also provides a product containing the polypeptide PGHFEAF.

[0033] In one embodiment, the product comprises food, medicine or health care product.

[0034] In one embodiment, the food comprises dairy products, fruit and vegetable products, meat products or soy products.

[0035] In one embodiment, the dosage form of the drug includes tablets, capsules, injections, liposome nanoparticles, sustained-release preparations, dispersible tablets or enteric-coated granules.

[0036] In one embodiment, the product also contains derivatives of the polypeptide PGHFEAF; the polypeptide derivatives refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain groups, amino terminus or carbonyl terminus of the polypeptide.

[0037] The present invention also provides the use of the polypeptide PGHFEAF in preparing a drug for sobering up and protecting the liver.

[0038] In one embodiment, the medicament further contains a pharmaceutically acceptable carrier.

[0039] In one embodiment, the content of the polypeptide PGHFEAF in the drug is at least 0.49 mM or 393.65 mg / g.

[0040] In one embodiment, the drug has both alcohol dehydrogenase activating activity and acetaldehyde dehydrogenase activating activity.

[0041] The present invention also provides the use of the polypeptide PGHFEAF in preparing a health product having an auxiliary protective effect on chemical liver damage.

[0042] Beneficial effects:

[0043] The present invention first identified a highly active ADH and ALDH activating polypeptide PGHFEAF from walnut protein, and its docking binding energy with ADH and ALDH was -8.2 and -10.5 kcal / mol, respectively. 50 The value was 0.49±0.09mM, ALDH EC 50 The value is 1.85±0.28mM; the polypeptide PGHFEAF is resistant to gastrointestinal digestion, and after 4h of in vitro digestion, the ADH and ALDH activation rates are retained by 58.43% and 41.58%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The effect of enzyme types on the activation rate of walnut peptide ADH and ALDH;

[0045] Figure 2 The activation rate of ADH and ALDH by walnut protein hydrolysate produced by alkaline protease;

[0046] Figure 3 Effects of simulated gastrointestinal digestion in vitro on the ADH and ALDH activation activities of walnut protein hydrolysates;

[0047] Figure 4 The molecular structure of PGHFEAF;

[0048] Figure 5 Molecular docking diagram of PGHFEAF and ADH;

[0049] Figure 6 Molecular docking diagram of PGHFEAF and ALDH;

[0050] Figure 7 Effect of PGHFEAF on ECs activated by ADH and ALDH 50 value;

[0051] Figure 8 Effects of simulated gastrointestinal digestion in vitro on the ADH and ALDH activation activities of PGHFEAF. DETAILED DESCRIPTION

[0052] The walnut kernels used in the following examples were purchased from Moore Farm Biotechnology Co., Ltd., and the commercial proteases used were purchased from Novozymes (China) Biotechnology Co., Ltd. The enzyme activity of neutral protease was 74898.8 U / mL, the enzyme activity of alkaline protease was 296816.9 U / mL, the enzyme activity of composite protease was 94187.9 U / mL, and the enzyme activity of flavor protease was 45268.1 U / mL.

[0053] The walnut protein powder used in the following examples is a dry protein powder after fat is removed.

[0054] The following examples involve a detection method for determining the activation rate of ADH and ALDH by substrate chemistry:

[0055] (1) Detection method of ADH activation rate

[0056] The detection principle is that ADH catalyzes the dehydrogenation of ethanol to produce acetaldehyde, while NAD + It is reduced to NADH. NADH has a maximum absorption peak at 340nm, while other components have no color. Therefore, the activity of ADH can be indirectly characterized by detecting the amount of NADH generated.

[0057] In a 96-well ELISA plate, 50 μL of sample was mixed with 150 μL of working solution (reagent 1: reagent 2: reagent 3 = 0.65: 0.05: 0.70), incubated at 37°C for 5 min, and then 50 μL of ADH solution (enzyme activity 0.2 U / mL) was added to stimulate the reaction, and the reaction was carried out at 37°C for 10 min. The absorbance was measured at 340 nm using an ELISA reader, and distilled water was used instead of the sample as a blank control. The ADH activation rate calculation formula is:

[0058]

[0059] (2) Detection method of ALDH activation rate

[0060] The detection principle is that acetaldehyde is oxidized by ALDH to produce NADH, and then the colorless probe is reduced to a colored product with strong absorbance at 450nm, so it can indirectly characterize the ALDH activity.

[0061] In a 96-well ELISA plate, 50 μL of sample was mixed with 200 μL of working solution (composed of acetaldehyde, NAD + The mixture was mixed with a mixture of a commercial probe and incubated at 37°C for 5 min, and then 50 μL of ALDH solution (enzyme activity of 0.5 U / mL) was added to stimulate the reaction, and the reaction was carried out at 37°C for 10 min. At the same time, distilled water was used instead of the sample as a blank control. The ALDH activation rate calculation formula is:

[0062]

[0063] In the following examples, the amino acid sequence of PGHFEAF is Pro-Gly-His-Phe-Glu-Ala-Phe.

[0064] Example 1: Method for obtaining walnut protein hydrolysate from walnut protein

[0065] (1) Extracting walnut protein from walnut kernels by alkali dissolution and acid precipitation

[0066] The defatted walnut powder was mixed with water at a ratio of 1:20 (w:v), the pH was adjusted to 10.00-11.0, and the mixture was stirred continuously for 3 hours at room temperature and then centrifuged (7000r / min, 15min), the supernatant was collected and stored, the pH of the supernatant was adjusted to 4.5 and then centrifuged again (7000r / min, 15min), the precipitate was redissolved, and the pH was adjusted to 7.0. After dialysis, the mixture was freeze-dried to obtain walnut protein powder.

[0067] (2) Producing walnut peptides by hydrolyzing walnut protein with protease

[0068] Walnut protein powder was mixed with water at a ratio of 1:25 (w:v) to prepare a protein solution with a substrate concentration of 4%. After mixing, the pH and temperature were adjusted to the optimal pH and temperature conditions of the enzyme used, and different proteases were added according to the addition amount of 10000U / g protein of enzyme substrate ratio for hydrolysis, and the hydrolysis was continued at room temperature with continuous stirring until the pH was constant. The hydrolysis conditions are shown in Table 1.

[0069] Table 1 Hydrolysis conditions of different proteases

[0070]

[0071] During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain a constant pH value of the solution.

[0072] After the hydrolysis was completed, the walnut protein hydrolysate was boiled for 10 minutes to inactivate the enzyme, cooled to room temperature, adjusted to pH 7.0, and centrifuged at 8000 r / min for 20 minutes. The supernatant was collected, dialyzed, and freeze-dried to obtain walnut protein hydrolysate powder under different protease hydrolysis conditions.

[0073] (3) The ADH and ALDH activation activities of walnut protein hydrolyzate powder obtained under different protease hydrolysis conditions were determined. The alkaline protease hydrolyzate with high activation activity was screened out ( Figure 1 ), the results showed that the activation rates of ADH and ALDH in alkaline protease hydrolysates were as high as 73.21±2.68 and 83.71±2.18%, respectively. 50The values ​​were 0.05546±0.0047 mg / mL and 0.0606±0.0096 mg / mL, respectively. Then the next step of sequencing was performed.

[0074] Example 2: In vitro simulated gastrointestinal digestion of walnut protein hydrolyzate powder

[0075] The specific steps are as follows:

[0076] Weigh 1 g of the alkaline protease hydrolysate obtained in Example 1 and dissolve it in 10 mL of 2000 U / mL pepsin hydrochloric acid solution (pH 3.0), and culture it at a constant temperature with shaking (37°C, 150 rpm, 2 h) to simulate gastric digestion (SGD). Then enter the simulated intestinal digestion (SID) stage, adjust the pH of the digestion system to 7.0 and make the volume to 20 mL, take a sample of 10 mL, quickly inactivate the enzyme and cool it, this time is recorded as 2 h. Add 100 U / mL trypsin to the remaining sample, take samples at different time points, inactivate the enzyme and quickly cool it. The freeze-dried samples were tested for ADH and ALDH activation activity. The results showed that ( Figure 3 ), after 2h of pepsin digestion, the ADH activation rate decreased from 73.61% to 68.25%, and the activity was retained at 92.72% (the activity retention rate is the ratio of the activation rate before and after digestion); the ALDH activation rate decreased from 83.71% to 60.24%, and the activity was retained at 61.04%. After in vitro simulated intestinal digestion, the final ADH and ALDH activation rates were 53.14% and 40.80%, respectively, still retaining a high activation activity.

[0077] Example 3: Method for separating and identifying ADH and ALDH activating peptides from walnut protein hydrolysate

[0078] The specific steps are as follows:

[0079] The specific implementation method is the same as that of Example 1, except that the protease is adjusted to alkaline protease to obtain walnut protein hydrolysate.

[0080] (1) Peptide sequence identification of walnut protein hydrolysates by peptidomics

[0081] The walnut protein hydrolyzate samples were first desalted using a C18 desalting column and then analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was an Orbitrap Exploris 480 mass spectrometer connected in series with an EASY-nanoLC 1200. A total of 2 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm×25 cm), and the sample was separated by a gradient of 60 min. The column flow was controlled at 350 nL / min, the column temperature was 40°C, the electrospray voltage was 2 kV, and the gradient started from 2.2% of phase B, increased to 50% in 51 minutes with a nonlinear gradient, increased to 90% in 3.5 minutes, and maintained for 5.5 minutes.

[0082] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 350-1500; resolution: 60,000; NormalizedAGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15,000; NormalizedAGC target: 50%; maximum injection time: 22 ms; collision energy: 30%; dynamic exclusion time: 30 s.

[0083] Tandem mass spectrometry analysis was performed using PEAKS Studio version 10.6. The uniprot_Cicerarietinum (version 202112, 24812 entries) database was searched using PEAKSDB. Protein card values ​​were: -10lgP ≥ 0, containing at least 1 unique peptide; peptide card values ​​were: -10lgP ≥ 20.

[0084] A total of 3554 peptides were identified after sequencing walnut protein hydrolysate using peptideomics.

[0085] (2) Using multiple peptide screening conditions, highly active ADH and ALDH activating peptides were screened from the 3554 peptides identified in (1), mainly including the following steps:

[0086] ① Preliminary screening conditions: Peptide Ranker software, protein database, Toxinpred software, and AllerTOP software were used to predict potential biological activity, protein source, toxicity, and allergenicity. From the sequenced peptides, those that were of protein origin, not modified by functional groups, and had a peak area of ​​1.00×10 6 , peptide sequences containing 3-12 amino acids and activity scores ≥ 0.7.

[0087] ② Rapid screening conditions: select peptide sequences with amphiphilicity, steric hindrance ≤0.65, hydrophobic amino acids ≥50% and characteristic amino acids ≥50%. Peptides with such characteristics have potential ADH activation activity.

[0088] (3) Screening and verification of peptides obtained from (2) using molecular docking

[0089] The specific steps are as follows:

[0090] ① Obtain small molecule ligands: Use Chem Draw (version ChemBio Draw 22.0.0) and Chem3D (version ChemBio Draw 22.0.0) software to construct the 2D and 3D structures of the peptide sequences to be docked.

[0091] ② Preparation of PDB files for ADH and ALDH: The protein crystal structures of ADH and ALDH (5ENV and 1O04, respectively) were retrieved from the protein database, and redundant ligands and water molecules were removed for subsequent docking with peptide segments.

[0092] ③Molecular docking: The format of ligand and receptor was converted to PDBQT in AutoDock Tools 1.5.7, and the molecular docking simulation experiment was performed using AutoDockVina software. The result was output as the predicted binding energy. The lower the binding energy, the stronger the binding between the ligand and the protein pocket. The ADH and ALDH activation peptides with published sequences in the prior art were excluded, and the remaining peptides were identified as potential ADH and ALDH activation peptides, which were synthesized in vitro by solid phase synthesis and their activation activity was verified.

[0093] Specifically, a total of 5 polypeptides were screened, and their binding energies with ADH and ALDH were all less than -7.5 kcal / mol (Table 2); the 5 polypeptides were synthesized in vitro by solid phase synthesis and their ADH and ALDH activation activities were measured.

[0094] Table 2 Docking binding energy of peptides and ADH molecules

[0095]

[0096] The five peptide segments after screening were synthesized by solid phase synthesis in the peptide chemical synthesis method, and the ADH and ALDH activation activities were determined by substrate chemical method to verify the screening results.

[0097] Table 3 ADH and ALDH activation activity of synthetic peptides

[0098]

[0099] In general, PGHFEAF has low binding energy with ADH and ALDH, but high binding degree. It also has strong in vitro activation ability. 50 The values ​​were 0.49±0.09mM and 1.85±0.28mM (Table 3), respectively, with strong activation ability. After conversion, the content of 0.49mM peptide in the solid product was 393.65mg / g. PGHFEAF was selected as the target peptide for this study. By consulting the BIOPEP-UWM database, it was found that PGHFEAF had not been reported in the literature. Therefore, PGHFEAF is a new bioactive peptide.

[0100] Example 4: ADH and ALDH Activating Peptide PGHFEAF

[0101] (1) Structural analysis of PGHFEAF

[0102] like Figure 4 As shown, the molecular structure of PGHFEAF was obtained by Chem Draw and Auto Dock.

[0103] (2) Molecular docking of PGHFEAF with ADH and ALDH

[0104] like Figure 5 As shown, PGHFEAF forms hydrogen bonds with eight amino acid residues in the ADH active pocket: HIS-171, ASN-166, THR-264, SER-289, ASN-262, ASN-107, and LYS-160. Figure 6 As shown, PGHFEAF forms hydrogen bonds with six amino acid residues, GLY-449, ALA-448, VAL-451, VAL-453, and SER-443, in the ALDH active pocket.

[0105] (3) Effect of PGHFEAFL on ECs activated by ADH and ALDH 50 value

[0106] The peptide sample PGHFEAF was prepared into solutions of different concentrations to determine its ADH and ALDH activation rates and draw a graph for analysis, and its ADH and ALDH half-inhibitory concentration (EC 50 ), that is, the peptide concentration required to reach 50% activation activity. Figure 7 As shown, the ADH EC of the peptide PGHFEAF 50 The value was 0.49±0.09mM, ALDH EC 50 The value is 1.85±0.28mM.

[0107] (4) PGHFEAFL simulates gastrointestinal digestion in vitro

[0108] Referring to the method of Example 2, PGHFEAF was subjected to in vitro simulated gastrointestinal digestion, and the changes in ADH and ALDH activation rates during the digestion process were measured. Figure 8 As shown in the figure, after 2h of pepsin digestion, the activation rates of ADH and ALDH of PGHFEAF decreased to 70.20% and 52.45%, respectively, and the activities were retained by 87.44% and 84.82%, respectively; after in vitro simulated intestinal digestion, the final activation rates of ADH and ALDH were 58.43% and 41.58%, respectively, indicating that PGHFEAF showed better digestibility resistance and biological stability. Among them, the activity retention rate is the ratio of the activation rate after digestion to the activation rate before digestion.

[0109] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A polypeptide PGHFEAF, characterized in that The amino acid sequence of the polypeptide is Pro-Gly-His-Phe-Glu-Ala-Phe.

2. A product containing the polypeptide PGHFEAF according to claim 1.

3. The product according to claim 2, characterized in that: The products include food, medicine or health products.

4. The product according to claim 3, characterized in that The food includes dairy products, fruit and vegetable products, meat products or bean products.

5. The product according to claim 4, characterized in that The dosage form of the drug includes tablets, capsules, injections, liposome nanoparticles, sustained-release preparations, dispersible tablets or enteric-coated granules.

6. The product according to claim 5, characterized in that The product also contains derivatives of the polypeptide PGHFEAF; the polypeptide derivatives refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino end or carbonyl end of the polypeptide.

7. Use of the polypeptide PGHFEAF according to claim 1 in the preparation of a drug for sobering up and protecting the liver.

8. The use according to claim 7, characterized in that In the drug, the content of the polypeptide PGHFEAF is at least 0.49 mM or 393.65 mg / g.

9. The use according to claim 8, characterized in that The drug has both alcohol dehydrogenase activating activity and acetaldehyde dehydrogenase activating activity.

10. Use of the polypeptide PGHFEAF according to claim 1 in the preparation of a health product having an auxiliary protective effect on chemical liver damage.

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